Characterization of Defects in Ion Transport and Tissue Development in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-Knockout Rats

Animal models for cystic fibrosis (CF) have contributed significantly to our understanding of disease pathogenesis. Here we describe development and characterization of the first cystic fibrosis rat, in which the cystic fibrosis transmembrane conductance regulator gene (CFTR) was knocked out using a pair of zinc finger endonucleases (ZFN). The disrupted Cftr gene carries a 16 base pair deletion in exon 3, resulting in loss of CFTR protein expression. Breeding of heterozygous (CFTR+/−) rats resulted in Mendelian distribution of wild-type, heterozygous, and homozygous (CFTR−/−) pups. Nasal potential difference and transepithelial short circuit current measurements established a robust CF bioelectric phenotype, similar in many respects to that seen in CF patients. Young CFTR−/− rats exhibited histological abnormalities in the ileum and increased intracellular mucus in the proximal nasal septa. By six weeks of age, CFTR−/− males lacked the vas deferens bilaterally. Airway surface liquid and periciliary liquid depth were reduced, and submucosal gland size was abnormal in CFTR−/− animals. Use of ZFN based gene disruption successfully generated a CF animal model that recapitulates many aspects of human disease, and may be useful for modeling other CF genotypes, including CFTR processing defects, premature truncation alleles, and channel gating abnormalities

Use of ferrets for electrophysiologic monitoring of ion transport

<div><p>Limited success achieved in translating basic science discoveries into clinical applications for chronic airway diseases is attributed to differences in respiratory anatomy and physiology, poor approximation of pathologic processes, and lack of correlative clinical endpoints between humans and laboratory animal models. Here, we discuss advantages of using ferrets (<i>Mustela putorus furo)</i> as a model for improved understanding of human airway physiology and demonstrate assays for quantifying airway epithelial ion transport <i>in vivo</i> and <i>ex vivo</i>, and establish air-liquid interface cultures of ferret airway epithelial cells as a complementary <i>in vitro</i> model for mechanistic studies. We present data here that establishes the feasibility of measuring these human disease endpoints in ferrets. Briefly, potential difference across the nasal and the lower airway epithelium in ferrets could be consistently assessed, were highly reproducible, and responsive to experimental interventions. Additionally, ferret airway epithelial cells were amenable to primary cell culture methods for <i>in vitro</i> experiments as was the use of ferret tracheal explants as an <i>ex vivo</i> system for assessing ion transport. The feasibility of conducting multiple assessments of disease outcomes supports the adoption of ferrets as a highly relevant model for research in obstructive airway diseases.</p></div

Measurement of lower airway potential difference (LAPD) in wild type ferrets.

<p><b>A</b>. Representative LAPD tracing in a sedated intubated ferret indicating PD changes following perfusion with Ringer’s solution, amiloride (100 μM), chloride-free, chloride-free + forskolin (20 μM), and CFTR-specific inhibitor GlyH101 (10 μM); N = 5. <b>B</b>: Summary tracing of PD changes in ferrets following infusion of the 5 sequential reagents, N = 5. <b>C</b>: Change in LAPD measurements from 5 (male and female) adult, wild type ferrets.</p

Potential difference measurements across nasal epithelium.

<p>Potential difference measurements across nasal epithelium.</p

Reproducibility of nasal potential difference (NPD) assays in ferrets.

<p><b>A</b>: Representative NPD tracing in ferrets one hour after acute intranasal administration with either vehicle (DMSO 0.001% in saline) or CFTR-specific inhibitor GlyH101 (10μM). <b>B</b>: Summary of CFTR-dependent PD differences in vehicle and GlyH101-treated ferrets. N = 4, * P<0.05.</p

Measurement of nasal potential difference (NPD) in wild type ferrets.

<p><b>A</b>: Representative NPD tracing in ferrets using the apparatus described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186984#pone.0186984.g001" target="_blank">Fig 1</a>. Under deep sedation, PD changes following perfusion with Ringer’s solution, Ringer’s solution with amiloride (100 μM), chloride-free solution, chloride-free with forskolin (20 μM), and chloride-free with CFTR-specific inhibitor GlyH101 (10 μM). <b>B</b>: Summary tracing of mean PD measurements (mean ± SEM) in ferrets following infusion of these 5 sequential reagents, N = 7. Ion channel activity of CFTR is quantified by PD changes in response to chloride-free + forskolin or by GlyH101 inhibitor. Similarly, ENaC activity is attributed to PD changes following amiloride treatment. <b>C</b>: Change in NPD (ΔPD) measurements from the same cohort of male and female adult, wild type ferrets plotted as individual measurements. <b>D</b>: Summary tracing of PD measurements (mean ±SEM) in male and female, adult, wild type ferrets acquired at two different times to validate reproducibility, N = 7. No discernable differences in ENaC or CFTR activity were found over 7 weeks. <b>E</b>: PD measurements of each animal subject plotted individually for each perfusate at two time points conducted 7 weeks apart.</p

Potential difference measurements across lower airway epithelium.

<p>Potential difference measurements across lower airway epithelium.</p

Schematic of laboratory set up for potential difference (PD) measurements in ferrets.

<p>Cartoon representing hardware and electrical connections representing PD apparatus modified from the human instrument recommended by the standard operating procedure of the CF Therapeutics Development Network and the European Clinical Trial Network [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186984#pone.0186984.ref019" target="_blank">19</a>]. Ground Lead: Connects electrical apparatus to ground for reference from which voltage is measured. Headstage: Connects electrode inputs to bioamplifier and safely isolates from wall AC current. Bioamplifier: Serves as voltmeter to measure PD. Analog-Digital Converter: Converts analog signals to digital form. K/Cl Calomel Electrodes: Measure PD. Agar Bridge: Connects positively charged (+) calomel electrodes to ferret nasal epithelium or lower airway epithelium through Airway Catheter and negatively charged (-) calomel electrodes to subcutaneous tissue through Butterfly Needle. Syringe Pumps: Deliver test solutions perfused sequentially from pump 1 to 5 with a flow rate of 4ml/hr (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186984#pone.0186984.s002" target="_blank">S1 Table</a>). The PD changes are observed and recorded on the computer using LabChart 7.0 software. Following the procedure, the nasal cannula and subcutaneous needle were removed, and anesthesia reversed with atipamezole (5 mg/kg body weight, IM) delivered at an equal volume as dexmedetomidine. All ferrets recovered within 10–15 min, and remained on a warming pad until aroused and moving. Supplemental oxygen was not required during the NPD procedures in our experience, however supplemental oxygen was delivered during recovery from anesthesia.</p

Measurement of short-circuit current (Isc) in cultured ferret bronchial epithelial cells and in explants of ferret trachea.

<p><b>A</b>: Representative hematoxylin and eosin stained image of a well-differentiated ferret bronchial epithelial cells grown at air-liquid interface. <b>B</b>: Representative Isc tracings of primary ferret bronchial epithelial cells sequentially exposed to forskolin (10 μM), followed by GlyH101 (20 μM) in the setting of amiloride (100 μM) and a chloride secretory gradient. <b>C</b>: Summary data of Isc measurements from different ALI cultures indicating stimulated Isc following acute addition of forskolin or GlyH101. N  =  4/condition. <b>D</b>: Representative Isc tracing of an <i>ex vivo</i> ferret trachea sequentially exposed to Ringer’s, chloride-free, forskolin (20 μM), followed by GlyH101 (20 μM) in the setting of amiloride (100 μM). <b>E</b>: Summary data of Isc measurements from different trachea showing stimulated Isc following acute addition of amiloride, forskolin, or GlyH101. N = 7/condition.</p